Published on Web 03/22/2006
Direct Hydrogen-Atom Abstraction by Activated Bleomycin: An Experimental and Computational Study Andrea Decker, Marina S. Chow, Jyllian N. Kemsley, Nicolai Lehnert, and Edward I. Solomon* Contribution from the Department of Chemistry, Stanford UniVersity, Stanford, California 94305 Received October 28, 2005; E-mail:
[email protected] Abstract: Bleomycin (BLM), a glycopeptide antibiotic chemotherapy agent, is capable of single- and doublestrand DNA damage. Activated bleomycin (ABLM), a low-spin FeIII-OOH complex, is the last intermediate detected prior to DNA cleavage following hydrogen-atom abstraction from the C-4′ of a deoxyribose sugar moiety. The mechanism of this C-H bond cleavage reaction and the nature of the active oxidizing species are still open issues. We have used kinetic measurements in combination with density functional calculations to study the reactivity of ABLM and the mechanism of the initial attack on DNA. Circular dichroism spectroscopy was used to directly monitor the kinetics of the ABLM reaction. These experiments yield a deuterium isotope effect, kH/kD ≈ 3 for ABLM decay, indicating the involvement of a hydrogen atom in the rate-determining step. H-atom donors with relatively weak X-H bonds accelerate the reaction rate, establishing that ABLM is capable of hydrogen-atom abstraction. Density functional calculations were used to evaluate the two-dimensional potential energy surface for the direct hydrogen-atom abstraction reaction of the deoxyribose 4′-H by ABLM. The calculations confirm that ABLM is thermodynamically and kinetically competent for H-atom abstraction. The activation and reaction energies for this pathway are favored over both homolytic and heterolytic O-O bond cleavage. Direct H-atom abstraction by ABLM would generate a reactive FeIVdO species, which would be capable of a second DNA strand cleavage, as observed in vivo. This study provides experimental and theoretical evidence for direct H-atom abstraction by ABLM and proposes an attractive mechanism for the role of ABLM in double-strand cleavage.
1. Introduction
Bleomycin (BLM, Figure 1), a glycopeptide antibiotic, is clinically used in cancer chemotherapy, especially against head, neck, and testicular cancers and Hodgkin’s disease.1-3 Its cytotoxic activity is believed to be due to its ability to carry out single- and especially double-strand cleavage of DNA in the presence of a reduced metal ion and dioxygen.4-8 The highest activity in ViVo is found for FeIIBLM.9 BLM provides five or six ligands for binding to the iron. Because no crystal structure exists, current knowledge of the Fe-BLM structure is based on spectroscopy,10-14 molecular dynamics modeling,15 density functional calculations,16,17 and (1) Umezawa, H. Anticancer Agents based on Natural Products;; Academic Press: New York, 1980. (2) Carter, S. K. Bleomycin Chemotherapy; Academic Press: New York, 1985. (3) Lazo, J. S.; Chabner, B. A. Cancer Chemotherapy and Biotherapy: Principles and Practice; Lippinkott-Raven: Philadelphia, PA, 1996. (4) Hecht, S. M. Acc. Chem. Res. 1986, 19, 383-391. (5) Stubbe, J.; Kozarich, J. W. Chem. ReV. 1987, 87, 1107-1136. (6) Stubbe, J.; Kozarich, J. W.; Wu, W.; Vanderwall, D. E. Acc. Chem. Res. 1996, 29, 322-330. (7) Burger, R. M. Chem. ReV. 1998, 98, 1153-1169. (8) Hecht, S. M. J. Nat. Prod. 2000, 63, 158-168. (9) Radtke, K.; Lornito, F. A.; Byrnes, R. W.; Antholine, W. E.; Petering, D. H. Biochem. J. 1994, 302, 655-664. (10) Loeb, K. E.; Zaleski, J. M.; Westre, T. E.; Guajardo, R. J.; Mascharak, P. K.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. J. Am. Chem. Soc. 1995, 117, 4545-4561. (11) Lehmann, T. E.; Ming, L. J.; Rosen, M. E.; Que, L. Biochemistry 1997, 36, 2807-2816. (12) Fulmer, P.; Zhao, C.; Li, W.; Derose, E.; Antholine, W. E.; Petering, D. H. Biochemistry 1997, 36, 4367-4374. 10.1021/ja057378n CCC: $33.50 © 2006 American Chemical Society
studies on BLM complexes with Co,18-21 Cu,22,23 and other metals.24-27 All studies agree on five nitrogen-based ligands (indicated in bold in Figure 1). The four equatorial ligands are generally accepted to be an imidazole nitrogen, a deprotonated amide, a pyrimidine nitrogen, and the secondary amine functionality of the β-aminoalanine. The primary amine of the β-aminoalanine has been proposed as an axial ligand. FeIIBLM reacts with dioxygen in the presence of reducing equivalents to form activated bleomycin (ABLM), the last intermediate detected prior to DNA strand scission.4,28 It is well (13) Loeb, K. E.; Zaleski, J. M.; Hess, C. D.; Hecht, S. M.; Solomon, E. I. J. Am. Chem. Soc. 1998, 120, 1249-1259. (14) Smolentsev, G.; Soldatov, A. V.; Wasinger, E. C.; Solomon, E. I. Inorg. Chem. 2004, 43, 1825-1827. (15) Lehmann, T. E. J. Biol. Inorg. Chem. 2002, 7, 305-312. (16) Freindorf, M.; Kozlowski, P. M. J. Phys. Chem. A 2001, 105, 7267-7272. (17) Karawajczyk, A.; Buda, F. J. Biol. Inorg. Chem. 2005, 10, 33-40. (18) Xu, R. X.; Nettesheim, D.; Otvos, J. D.; Petering, D. H. Biochemistry 1994, 33, 907-916. (19) Lui, S. M.; Vanderwall, D. E.; Wu, W.; Tang, X. J.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. J. Am. Chem. Soc. 1997, 119, 9603-9613. (20) Lehmann, T. E.; Serrano, M. L.; Que, L. Biochemistry 2000, 39, 38863898. (21) Fedeles, F.; Zimmer, M. Inorg. Chem. 2001, 40, 1557-1561. (22) Sugiyama, M.; Kumagai, T.; Hayashida, M.; Maruyama, M.; Matoba, Y. J. Biol. Chem. 2002, 277, 2311-2320. (23) Lehmann, T. E. J. Biol. Inorg. Chem. 2004, 9, 323-334. (24) Mouzopoulou, B.; Kozlowski, H.; Katsaros, N.; Garnier-Suillerot, A. Inorg. Chem. 2001, 40, 6923-6929. (25) Papakyriakou, A.; Katsaros, N. Eur. J. Inorg. Chem. 2003, 3001-3006. (26) Papakyriakou, A.; Mouzopoulou, B.; Katsaros, N. J. Biol. Inorg. Chem. 2003, 8, 549-559. (27) Papakyriakou, A.; Bratsos, I.; Katsarou, M.; Katsaros, N. Eur. J. Inorg. Chem. 2004, 3118-3126. J. AM. CHEM. SOC. 2006, 128, 4719-4733
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Figure 1. Structure of bleomycin (likely metal ligating atoms indicated in bold blue).
established by electron paramagnetic resonance (EPR) spectroscopy,28-30 Mo¨ssbauer spectroscopy,31 electron nuclear double-resonance (ENDOR) spectroscopy,30,32 mass spectrometry,33 X-ray absorption spectroscopy (XAS),34 and magnetic circular dichroism (MCD) spectroscopy35 that ABLM is a lowspin (ls) ferric-hydroperoxo complex, BLM-FeIII-OOH. ABLM can be formed when ferrous BLM, (BLM)FeII-OH2, reacts with O2 or when ferric BLM, (BLM)FeIII-OH, reacts with excess H2O2.28,36 Both pathways result in the same ABLM species,28 and both generate the same DNA cleavage products.36,37 In the absence of DNA, the decay of ABLM yields FeIIIBLM and water as final products.28 The rate of ABLM decay is known to be first order in the concentration of ABLM. The half-life for the decay of ABLM has been measured to be ∼1.5 min at 0 °C in buffer (50 mM sodium cacodylate, pH 7.2)37 and ∼2 min at 4-6 °C in unbuffered solution.28 To understand the mechanism of ABLM decay, several studies have analyzed the decay products for reactive oxygen species, however with different results: one study did not detect peroxides (H2O2) or superoxides (O2•-),38 another identified small amounts of peroxides,39 and a third observed only small amounts of hydroxy radicals (•OH) and superoxides.40 The fluorescence of the BLM bithiazole tail was observed to be altered upon ABLM decay.41 Kinetic studies of the formation (28) Burger, R. M.; Peisach, J.; Horwitz, S. B. J. Biol. Chem. 1981, 256, 1163611644. (29) Sugiura, Y. J. Am. Chem. Soc. 1980, 102, 5208-5215. (30) Veselov, A.; Sun, H.; Sienkiewicz, A.; Taylor, H.; Burger, R. M.; Scholes, C. P. J. Am. Chem. Soc. 1995, 117, 7508-7512. (31) Burger, R. M.; Kent, T. A.; Horwitz, S. B.; Mu¨nck, E.; Peisach, J. J. Biol. Chem. 1983, 258, 1559-1564. (32) Veselov, A.; Burger, R. M.; Scholes, C. P. J. Am. Chem. Soc. 1998, 120, 1030-1033. (33) Sam, J. W.; Tang, X.-J.; Peisach, J. J. Am. Chem. Soc. 1994, 116, 32505256. (34) Westre, T. E.; Loeb, K. E.; Zaleski, J. M.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. J. Am. Chem. Soc. 1995, 117, 1309-1313. (35) Neese, F.; Zaleski, J. M.; Loeb Zaleski, K.; Solomon, E. I. J. Am. Chem. Soc. 2000, 122, 11703-11724. (36) Kuramochi, H.; Takahashi, K.; Takita, T.; Umezawa, H. J. Antibiot. 1981, 34, 576-582. (37) Natrajan, A.; Hecht, S. M.; van der marel, G. A.; van Boom, J. H. J. Am. Chem. Soc. 1990, 112, 3997-4002. (38) Burger, R. M.; Horwitz, S. B.; Peisach, J.; Wittenberg, J. B. J. Biol. Chem. 1979, 254, 12299-12302. (39) Oberley, L. W.; Buettner, G. R. FEBS Lett. 1979, 97, 47-49. (40) Sugiura, Y.; Kikuchi, T. J. Antibiot. 1978, 31, 1310-1312. (41) Nakamura, M.; Peisach, J. J. Antibiot. 1988, 41, 638-647. 4720 J. AM. CHEM. SOC.
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and decay of ABLM have been challenging, since ABLM does not have a distinguishable spectroscopic absorption feature that can be easily monitored. Past studies involved freeze-quench methods to monitor changes to the EPR spectrum of ABLM, or the measurement of DNA cleavage ability of ABLM at set time points.28,42 There are two major DNA binding interactions for BLM. The bithiazole tail binds nonspecifically via partial intercalation43,44 or through interactions with the minor grove.45 Hydrogenbonding interactions between the pyrimidine in the metal binding domain and the DNA guanine are thought to be responsible for the sequence specificity of DNA cleavage.46-49 DNA strand scission is initiated through the abstraction of the C-4′ hydrogen atom of the backbone deoxyribose sugar50,51 mainly at pyrimidine nucleotides of preferred sites containing 5′-GC and 5′-GT sequences.52,53 After this initial attack, which generates a radical centered on the sugar C-4′, DNA cleavage can proceed via two pathways,54-59 producing free bases independent of O2 and base propenals when additional O2 is available. Although considerable research has been devoted to the reactivity of bleomycin, the nature of the active oxidizing species attacking the sugar C-4′ and the mechanism of this C-H bond cleavage reaction are still open issues. Three possible reaction pathways for the reactivity of ABLM in hydrogen-atom abstraction from DNA (Scheme 1) have been invoked: (1) Heterolytic cleavage of the O-O bond, generating a catalytically active “FeVdO” species, as has been observed in heme systems, where the formally “FeVdO” is stabilized by oxidation of the porphyrin ligand (Por), leading to a (Por+•)FeIVdO species (“compound I”). (2) Homolytic O-O bond cleavage, leading to a reactive FeIVdO intermediate and OH radical, as has been shown to occur for low-spin FeIIIOOH/R model systems and has been suggested to occur in mechanistic cycles of models of nonheme iron enzymes. (3) H-atom abstraction from the C-4′ of the DNA deoxyribose sugar directly by the hydroperoxo complex, ABLM. (42) Burger, R. M.; Tian, G.; Drlica, K. J. Am. Chem. Soc. 1995, 117, 11671168. (43) Povirk, L. F.; Hogan, M.; Dattagupta, N. Biochemistry 1979, 18, 96-101. (44) Wu, W.; Vanderwall, D. E.; Stubbe, J.; Kozarich, J. W.; Turner, C. J. J. Am. Chem. Soc. 1994, 116, 10843-10844. (45) Abraham, A. T.; Zhou, X.; Hecht, S. M. J. Am. Chem. Soc. 2001, 123, 5167-5175. (46) Wu, W.; Vanderwall, D. E.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. J. Am. Chem. Soc. 1996, 118, 1281-1294. (47) Vanderwall, D. E.; Lui, S. M.; Wu, W.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. Chem. Biol. 1997, 4, 373-387. (48) Boger, D. L.; Ramsey, T. M.; Cai, H.; Hoehn, S. T.; Kozarich, J. W.; Stubbe, J. J. Am. Chem. Soc. 1998, 120, 53-65. (49) Kemsley, J. N.; Zaleski, K. L.; Chow, M. S.; Decker, A.; Shishova, E. Y.; Wasinger, E. C.; Hedman, B.; Hodgson, K. O.; Solomon, E. I. J. Am. Chem. Soc. 2003, 125, 10810-10821. (50) Wu, J. C.; Kozarich, J. W.; Stubbe, J. Biochemistry 1985, 24, 7562-7568. (51) Worth, L.; Frank, B. L.; Christner, D. F.; Absalon, M. J.; Stubbe, J.; Kozarich, J. W. Biochemistry 1993, 32, 2601-2609. (52) Takeshita, M.; Grollman, A. P.; Ohtsubo, E.; Ohtsubo, H. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 5983-5987. (53) D’Andrea, A. D.; Haseltine, W. A. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 3608-3612. (54) Haidle, C. W.; Weiss, K. K.; Kuo, M. T. Mol. Pharmacol. 1972, 8, 531537. (55) Povirk, L. F.; Wu¨bker, W.; Ko¨hnlein, W.; Hutchinson, F. Nucleic Acids Res. 1977, 4, 3573-3580. (56) Povirk, L. F.; Ko¨hnlein, W.; Hutchinson, F. Biochim. Biophys. Acta 1978, 521, 126-133. (57) Burger, R. M.; Berkowitz, A. R.; Peisach, J.; Horwitz, S. B. J. Biol. Chem. 1980, 255, 11832-11838. (58) Giloni, L.; Takeshita, M.; Johnson, F.; Iden, C.; Grollman, A. P. J. Biol. Chem. 1981, 256, 8608-8615. (59) Breen, A. P.; Murphy, J. A. Free Radical Biol. Bed. 1995, 18, 10331077.
Direct H-Atom Abstraction by Activated Bleomycin Scheme 1 . Possible Reaction Pathways for ABLM
Pathway 1 is reminiscent of the activation and mechanism of the heme enzyme cytochrome P450 and has been proposed due to the strong similarity between the chemistry of ABLM and that of P450 (i.e., H-atom abstraction from unactivated C-H bonds, hydroxylation, and epoxidation).60-63 However, a detailed quantum chemical study on the electronic structure of ABLM and its reactivity35 suggested strongly that heterolytic O-O bond cleavage is energetically unfavorable, less favorable for BLM than for heme systems by at least 70 kcal/mol.64 Heterolytic cleavage would lead to a product which is best described as a (BLM•+)FeIVdO with the hole localized on the deprotonated amide, in contrast to the heme compound I species, where the hole is delocalized over the porphyrin ring. Ligand-to-metal charge-transfer (LMCT) spectra show experimentally that it is energetically much harder to remove an electron from the bleomycin ligand than from the porphyrin ring,35,65 making heterolytic O-O bond cleavage in BLM energetically much less favorable. Pathway 2 was evaluated by studying the electronic structures and reactivities of ls ferric-alkylperoxo66 and -hydroperoxo67 model complexes. The bonding of ls FeIII-OOH is characterized by strong Fe-O and weak O-O bonds. This species is thermodynamically and kinetically competent to undergo homolytic cleavage of the O-O bond to yield FeIVdO and •OH. DFT studies of the reaction mechanism show no additional activation barrier along the reaction coordinate.68 A parallel study on the homolytic cleavage of ABLM gave similar results: Theoretically, ABLM is thermodynamically and kinetically competent to homolytically cleave the O-O bond with a calculated reaction free energy of ∆G ) 13 kcal/mol and no additional kinetic activation barrier.67 However, the fact that initial attack occurs through specific hydrogen-atom abstraction only from the C-4′ on the deoxyribose sugar,69,70 with an isotope selection effect kH/kD ) 2-7,51 (60) Padbury, G.; Sligar, S. G. J. Biol. Chem. 1985, 260, 7820-7823. (61) Murugesan, N.; Hecht, S. M. J. Am. Chem. Soc. 1985, 107, 493-500. (62) Heimbrook, D. C.; Carr, S. A.; Mentzer, M. A.; Long, E. C.; Hecht, S. M. Inorg. Chem. 1987, 26, 3835-3836. (63) Ehrenfeld, G. M.; Murugesan, M.; Hecht, S. M. Inorg. Chem. 1984, 23, 1496-1498. (64) Solomon, E. I.; Decker, A.; Lehnert, N. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3589-3594. (65) Cheesman, M. R.; Greenwood, C.; Thomson, A. J. AdV. Inorg. Chem. 1991, 36, 201-255. (66) Lehnert, N.; Ho, R. Y. N.; Que, L., Jr.; Solomon, E. I. J. Am. Chem. Soc. 2001, 123, 8271-8290. (67) Lehnert, N.; Ho, R. Y. N.; Neese, F.; Que, L., Jr.; Solomon, E. I. J. Am. Chem. Soc. 2002, 124, 10810-10822. (68) Lehnert, N.; Ho, R. Y. N.; Que, L., Jr.; Solomon, E. I. J. Am. Chem. Soc. 2001, 123, 12802-12816. (69) Wu, J. C.; Kozarich, J. W.; Stubbe, J. J. Biol. Chem. 1983, 258, 46944697.
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argues against an involvement of the very reactive and unselective hydroxyl radical (produced in homolytic O-O bond cleavage). A large tritium selection effect observed for the reaction of ABLM with a DNA model poly(dA-dU) provides evidence for the involvement of C-H bond cleavage in the ratedetermining step.50 On the other hand, 18O isotope studies42 demonstrate that the O-O bond cleavage is also involved in the rate-determining step. The proposed third pathway suggests that ABLM itself is the active oxidizing species which directly abstracts the hydrogen atom. A comparison of the reaction energies of the three proposed pathways for ABLM shows that the reaction energies for homolytic and heterolytic cleavage are comparable and endergonic (∆G ≈ 13 kcal/mol). The direct H-atom abstraction is exergonic (∆G ) -7 kcal/mol) and thus thermodynamically favored.64 The present investigation extends these studies on the reactivity of ABLM and evaluates with experimental and computational methods the kinetics of a direct H-atom abstraction reaction by ABLM. Since ABLM does not have a characteristic absorption feature that can be easily monitored, kinetic studies of ABLM decay have been challenging. However, using circular dichroism (CD) spectroscopy, we have identified such a feature and have measured the kinetics of ABLM decay and its reaction with hydrogen-atom donor substrates through deuterium isotope studies. Furthermore, density functional calculations were used to map the twodimensional potential energy surface (PES) for the direct hydrogen-atom abstraction reaction by ABLM (one dimension being the cleavage of the O-O bond, the second dimension the transfer of the hydrogen atom). In addition, we propose a mechanism for initiating the second strand cleavage event and contrast the direct H-atom abstraction pathway to the heterolytic O-O bond cleavage pathway. This study provides new insight into the reaction mechanism of ABLM on a molecular level and the effectiveness of this anti-cancer drug in DNA cleavage. 2. Methods 2.1. Experimental Procedures. 2.1.1. FeIIBLM. Solutions of ferrous bleomycin were prepared under strict anaerobic conditions. Bleomycin sulfate (Calbiochem) was evacuated and back-filled with argon 20 times in a Teflon-capped conical vial. FeII(NH4)2(SO4)2‚6H2O (FeIIAS, J.T. Baker) was purged with argon for 2 h. HEPES buffer (100 mM, Sigma) was prepared in deionized water and adjusted to pH 7.8 with NaOH. Deionized water and HEPES buffer were degassed by 5-10 freezepump-thaw cycles at 10-3 Torr in Teflon- and ground-glass-stoppered flasks. All degassed materials were immediately placed into a Vacuum Atmospheres Nexus-1 anaerobic glovebox (
